Trans fatty acids in infantile nutrition

NUTRITION RESEARCH, Vol. 13, Suppl. 1, pp. $47-$59, 1993 0271-5317/93 $6.00 + .00 Printed in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.

TRANS FATTY ACIDS IN INFANTILE NUTRITION B. Berra 1 Institute of General Physiology & Biological Chemistry, School of Pharmacy, University of Milano, Milano, Italy.

ABSTRACT Trans fatty acids arise from hydrogenation in vivo and in vitro. Estimates of dietary intake of trans acids are available from a few countries and show an enormous variation from one population group to another and even within one food type. The impact of trans fatty acid consumption on cell membrane composition, growth and development, risk of cardiovascular disease, and occurrence of cancer is not clear. Published data have several inconsistencies and are open to different interpretation. Given the complexity of the situation, it is prudent to avoid making sweeping recommendations until more specific data are available. However, large intakes of trans fatty acids should be avoided. KEY WORDS: Trans fatty acids, Hydrogenation, Growth, Dietary guidelines, Cardiovascular disease, Cancer.

INTRODUCTION Trans isomers of unsaturated fatty acids are formed during hydrogenation processes both in ruminant animals (bio-hydrogenation by bacteria) and in oil refinery in order to change the physical properties of oils, i.e. to increase their solid content and their melting point; indeed partially hydrogenated fats result in a better texture and stability (1). The range of isomers formed in the two processes is similar and in one sense therefore the chemical hydrogenation still produces only "natural" products. However, the content of trans acids in partially hydrogenated oils is usually much higher than that found in milk and in animal fats. ESTIMATES OF DIETARY INTAKE OF TRANS ACIDS Repeated concern has been expressed over the possible consequences to health brought 1Correspondence to: Dr. B. Berra, Institute of General Physiology & Biological Chemistry, School of Pharmacy, University of Milano, Milano, Italy.

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about by the increased consumption of trans fatty acids in recent years, due to the so called "cholesterol phobia" (2) which resulted in the replacement of butter or other animal fats with the "cholesterol free" solid vegetable oils. The average amounts currently eaten in countries where hydrogenated fats occur widely in food (which is not yet the case in Italy) have been estimated as at least 4 % of total energy (3) and possibly two to three times that (4). An attempt to calculate the daily intake of trans fatty acids is met with many difficulties. Only approximate values can be calculated; an example are the data reported in table 1 based on the figures available in the literature. The trans fatty acids content of different food consists mainly of monoenes, but dienes and trienes are also present. Their value is generally expressed as a percentage of total fatty acids, as indicated in Table 2 adapted, from Enig et al. (11) This table shows the extent to which the amount of trans fatty acids may vary, not only from one food to another, but also from one sample of a given food to another; moreover it turns out that the trans acid content in the diet is not only due to margarines but also to different processed food products containing partially hydrogenated oils used as shortenings. It must be taken into account that, as previously indicated, trans acids can be found in animal fats. Feed stuffs used in the rations of farm animals contain an average 3-6 per cent fat, generally high in polyunsaturated fatty acids (PUFA). Yet butter and animal fats, in general, are low in PUFA, because both the biosynthesis of fatty acids in the animal body and the partial biohydrogenation taking place in the rumen of cattle and other ruminants always yield saturated and monounsaturated fatty acids.

Table 1 Recent Estimates of Daily Intake of Trans Acids Year

Country

Average (g)

1980 1985 1990 1987 1983 1989 1981 1984

U.S.A. U.S.A. U.S.A. U.K. Finland Finland Holland Sweden

7.55 10.00 12.3-13.3 7.00 5.6 1.5-1.9 17.00 7.00

Maximum

28 27

23.8

Reference 5 6 4 7 8 8 9 10

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Table 2 Total Trans Fatty Acid Content of Some Foods (Percent of Total Fatty Acids) Foods

Total traits fatty acids

Bread and roils Cakes Crackers French fries Instant and canned puddings Stick margarines Soft margarines Shortenings Snack chips and pretzels Butter

10.4 to 27.9 10.1 to 24.0 2.8 to 31.6 4.6 to 35.1 30.5 to 36.1 18.0 to 36.0 11.2 to 21.3 13.0 to 37.3 14.4 to 33.4 <0.1 to 1.2

The enzymes of the rumen microflora responsible for biohydrogenation include isomerases. These enzymes can produce a certain number of trans fatty acids, for example vaccenic acid, one of the most important fatty acids so produced. It has a chain length of 18 carbon atoms with one double bond on carbon 11 from the carboxyl group; it is a geometrical and positional isomer of oleic acid (cis-C18:lA9) and its formula is trans-C18:l All. Vaccenic acid represents 75 per cent of the trans isomers of oleic acid in the rumen content where trans dienes and trienes are only found in traces (12). Vaccenic acid, like other isomers formed by biohydrogenation in the rumen, may be absorbed by the intestine, circulated in the blood, and ultimately found in meat and milk fat. According to of the literature, the trans fatty acid content of butter is probably less than 2.5 percent (13, 14). In conclusion, in our day-to-day life, less than 5 per cent of total trans fatty acids consumed is supplied by animal fats; partially hydrogenated vegetable oils used in the preparation of numerous foods on the other hand supply more than 95 per cent. Trans isomers of fatty acids in human milk and infant foods Whereas isomeric fatty acids may have no effect on an adult, in theory infants may be particularly vulnerable to the effects of factors that can interfere with essential fatty acid metabolism and normal membrane structure. Thus, it is important to know the trans fatty acid content of food consumed during this period of rapid development. Trans fatty acids comprise 2-5% of total fatty acids in human milk. The amount of transoctadecenoic acid (18:lt) appearing in the milk reflects the trans acid content of the maternal diet consumed on the previous day (15). Infant formulas generally contain lower amounts of trans fatty acids, with values of 0.1-2.0% of total fatty acids reported. One brand of infant formula analyzed by Hanson and Kinsella (16) contained 15.7% of the fatty acids as trans isomers, but all the others contained less than 2 %.

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Table 3 Trans Fatty Acid Content of Human Milk and Infant Foods*

Food itema Human milk (6) Human milk (3) Human milk (1) Human milk (8) Infant formula (3) Infant formula (11) Infant formula (1) Baby food (3) Dry infant cereal (8)

Trans fatty acids (•)b Min. Max. Avg. 3.18 2.1

5.43 4.0

0.1 0.8

1.3 2.0

0.2 0.3

7.6 0.6

4.14 3.1 tr 4.76 0.5 1.2 15.7 3.2 0.5

Fat (wt%)

3.8

6.0 7.0 5.82 3.5

aNumber of subjects/brand analyzed appears in parentheses following food item. bpercent of total fatty acids. *Adapted from M.C. Craig Schmidt in: Fatty acids in food and their health implication. C. Kuang Chow, ed, Marcel Dekker, 1992: 363-398.

Three types of baby foods were analyzed for trans fatty acid content by Slover and Lanza (17), and eight brands of dry infant cereals by Hanson and Kinsella (16). All of these products except the lamb broth (17) contained less than 2% trans fatty acids (Table 3). Thus, foods normally consumed by infants contain relatively low levels of trans fatty acids. In most cases trans isomeric fat in these products is no greater than that found in cow's milk.

METABOLIC FATE AND BIOCHEMISTRY OF TRANS FATTY ACIDS The major reason for the concern about trans fatty acids was the realization that a trans double bond alters the conformation of the fatty acid structure; in fact the trans isomers tend to have a "straighter" structure than their cis counterparts, which is responsible for their higher melting points. This structure suggests that the trans isomers are more like saturated fatty acids. Because these isomers have different configurations, it is conceivable that the trans bond has an effect on the binding constants of the enzymes involved in fatty acid metabolism. In this field, much research was done in animal or in vitro models, while in vivo studies in man were concerned mainly with the analysis of blood lipids.

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ABimal models and in vitro experiments a) General parameters Aaes-Jorgensen and Dam (18) found that increasing amounts of severely hydrogenated groundnut, whale, herring or seal oil as the only fat source in the diet caused a progressive decline in the growth of rats. These results were confirmed by Gottenbos (19) who attributed the effect of hydrogened fats to a deficiency in essential fatty acids. In our laboratory we found that the retarded growth of rats fed elaidic rich diet was simply due to a decrease of food consumption by the animals (20). Subsequently in a large number of studies in different animal species (rat, mouse, rabbit, swine, monkey) fed diets containing variable but always relevant quantities of trans fatty acids or partially hydrogenated vegetable oils, and provided with an adequate amount of EFA, no problems associated with growth, organ size or development, reproduction, tumor or longevity were detected. Histological examination of organs of such animals found no abnormalities or increased incidence of atherosclerotic plaques (21).

b) Biochemical studies The three most important functions of fatty acids are related to: i) energy supply and storage; ii) biomembrane composition and function; iii) biosynthesis of metabolic regulators (prostaglandins, thromboxanes, leukotrienes): this function is restricted to EFA. Digestibility and absorption Studies were done either with partially hydrogenated fats or with triglycerides containing elaidic acids and/or trans dienes (22-24). It was concluded that the trans fatty acids most commonly found in partially hydrogenated vegetable oil are digested and absorbed as well as their cis isomers which occur naturally in the parent oils before partial hydrogenation. Utilization as energy source Fatty acids are used as a source of energy either directly or after storage in adipose tissue. The energy of fatty acids is released by g-oxidation. Early in vivo animal experiments (22,23,25,26) have demonstrated that the overall oxidation rate of trans fatty acids is comparable to that of other fatty acids. Where trans dienes are concerned the oxidation is complete and leaves no residues of unsaturated short-chain molecules (27). With in vitro experiments it was shown also that isolated mitochondria metabolize both cis and trans fatty acids by the same mechanism but with some differences in their rate of oxidation, which is faster for the cis isomers. Moreover the rate increases, at least in the case of monoenes, as the double bond moves away from the carboxyl group. As a consequence vaccenic acid, a trans isomer produced by biohydrogenation, is catabolized at a rate closer to that of oleic acid, a natural cis isomer, than to that of elaidic acid (28). It was assumed that the limiting factor was not the activity of the enzyme systems

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involved in the/3-oxidation pathway, but rather the lower speed with which the trans fatty acids, as compared to the cis isomers, are transported through the inner mithocondrial membrane (29). Biomembrane comoosition Fatty acids constitute an integral, structural pan of complex lipids-phospholipids and cholesterol esters, which are considered the backbone of biomembrane. It is known that the physicochemical properties of these compounds and of the membrane itself are markedly influenced by the nature of their component fatty acids and their proportion and distribution, which on the other hand is correlated to the fatty acid composition of the diet. Fatty acids occur in phospholipids in specific positions according to their degree of unsaturation: the most highly unsaturated are esterifled to the 2 - position - where arachidonic acid is specifically present whereas in the 1 position saturated and monoenoic fatty acids are preponderant. In animals trans monoenoic and polyenoic acids in all the tissues except the brain (30) are incorporated into phospholipids preferably at the 1 position as if they were saturated fatty acids. The result is that both saturated and trans unsaturated fatty acids are incorporated in the 1 position leaving more opportunity for the cis polyunsaturated fatty acids to occupy the 2 position. Consequently the phospholipids synthesized following high intake of trans fatty acids would be unsaturated to a greater degree than if the natural cis isomers were consumed (31). Kummerow (32) did not agree with this conclusion stating that "the polyunsaturated fatty acid have a kinked structure; if such an acid is replaced by a trans one, the kink is removed and the structure becomes straighter". This means, to my opinion, more rigid. Whether these changes in membrane fluidity influence the functions of the membrane and/or the activities of the associated enzymes is still unclear; however the oxidative phosphorylation process which take place in the inner mitochondrion membrane is not modified by the incorporation in the membrane phospholipids of trans fatty acids (33). Metabolism of essential fatty acids and prostanoid biosynthesis The diet must supply a certain amount of polyunsaturated fatty acids (PUFA) particularly linoleic, a-linolenic and arachidonic acid. The last one is also produced by elongase and desaturase enzymes acting on the precursor linoleic fatty acid. The differences in methodological approaches (in vitro or in vivo systems; utilization of partially hydrogenated vegetable oils, or of synthetic formula containing different amount of trans isomers, presence or absence of EFA) created a great deal of confusion. The main controversies concern: (i) the synthesis of arachidonic acid and its utilization as a precursor for prostainoid production. Gottenbos (19) concluded that the conversion of linoleic acid to arachidonate is affected only by excessive amounts oftrans-9, trans 12-octadecadienoic acid. Moreover according to this author trans fatty acids or their derivatives are not incorporated in the 2-position of phospholipids when PUFA of the linoleic acid family are available. Therefore interference in the synthesis of prostanoids seems unlikely. Applewhite (34) and Zevenbergen (33) agreed with these conclusions while on the contrary Kinsella (35) found that blood platelets of those animals fed on trans, trans linoleic acid contained less arachidonic acid and less PGE 2 and PGF 2 activity

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than those fed on cis, cis linoleic acid; (ii) the activity of elongase and desaturase which are involved in the metabolism of polyenoic fatty acids. Mahfouz et al (36,37) and Kinsella et al. (35) have shown that trans 18:1 can be converted to cis,trans 18:2 and cis,cis 18:2 isomers which could be the precursor of new or unusual long-chain polyunsaturated fatty acids. The latter in turn, could be precursor of unnatural series of prostanoid derivatives having biological activities different from the normal ones. This concern is supported by Holman et al. (38) and by Emken (39). On the contrary Gottenbos (19) and Lemarchal (40) have shown that trans fatty acids are not or little metabolized by desaturase and elongase enzymes nor did they influence their activity. The effect of trans fatty acids on the metabolism of EFA and on prostanoids biosynthesis, which are of crucial interest for organ metabolism, needs to be further clarified.

Human studies As reported in the introduction, the main in vivo human studies are related to the incorporation of trans fatty acids in serum lipids (and in some cases in tissue lipids) and in plasma lipoproteins and to their effect on plasma cholesterol concentration. (a~. Digestion and absorntion of trans isomer containing fats. Direct and indirect evidences based on animal experiments and on the detection of isomeric fatty acids in human tissues indicate that these isomers are well absorbed (41). Lack of symptoms, such as diarrhea, in subjects fed hydrogenated vegetable oils is also an indirect evidence that these fats are normally digested and absorbed. As a matter of fact, many of the data found by Emken (42) were calculated from chylomicron triglyceride analysis. Also the absorption of the even-numbered trans positional isomers is normal despite their melting points of about 50~ which is well above body temperature (21). These results are consistent with data indicating that pancreatic lipase activity is not sensitive to the trans fatty acid configuration (43). Co). Incorporation into plasma and lipoprotein lipids. All different C18:1 isomers contained in dietary triglycerides are uptaken by chylomicrons and transported through the lymphatic system to the blood stream similarly as non-hydrogenated fats. On the contrary, the incorporation, as well as removal, of the various isomers into plasma triglycerides (total TG of chylomicrons, VLDL, LDL and HDL) is selective depending on the double bond position and configuration. These characteristics also influence the activity of cholesterol acyl transferase and hence the esterification of cholesterol, indicating the preferential incorporation of cis isomers in this molecule. As far as plasma phospholipids are concerned the data from Emken (19) indicate that trans isomers preferentially occupy the 1 position of glycerol (as saturated fatty acids usually do) while natural cis isomers esterify the OH-group at the 2-position. Also the distribution of C 18:1 isomers in lipoprotein classes indicates that isomeric fatty acid metabolism is influenced by the type of lipoprotein as well as by the lipid which is present in the lipoprotein itself. {c). Incorporation into tissue lipids. Review of the data from the literature reported by Ohlrogge (44) indicates that the trans isomer levels in different human tissue (as percentage of

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total fatty acids) are below 10% and average values usually fall below 5%. In the cases when the determinations were made either on atheroma or on tissues from subjects who died from atherosclerosis, no correlation could be found between tissue trans acid percentage and cause of death. In all subjects and in all lipid classes examined by Ohlrogge triglycerides contained higher levels of trans than any of the individual phospholipids, sharply in contrast with the data from animal models. This observation may indicate that: (i) A greater proportion of fatty acids in phospholipids originates from de novo biosynthesis or more probably discrimination occurs against incorporation of unusual isomers into phospholipids. (ii) - the trans isomers are preferentially oxidized rather than used in the formation of structural lipids. This point is supported also by the findings that in no cases the above mentioned author could observe major accumulation of a given isomer similar to that observed in rats fed on diets high in hydrogenated fats. Human metabolism appears able to turn over by oxidation these unusual isomers at rates sufficient to prevent their accumulation in the tissue lipids. However more recent data (G. Hornstra, personal communication) seem to demonstrate that, also in humans, trans fatty acids are incorporated into membrane phospholipids. (d). Oxidation. Since partially hydrogenated vegetable oils supply approximately 6% of the total fat in the U.S. diet (5) the low trans content of human tissue lipids is an indirect evidence for preferential oxidation. This assumption received a further confirmation by the data found in the analysis of human heart lipids which demonstrate that trans isomers of C 18:1 are present at lower level than in hydrogeneted oils or adipose tissue lipids (45). Moreover, in contrast to in vitro rat heart data, homogenized human heart tissue osidizes trans C 18:1 at nearly the same rate as the corresponding natural cis counterpart, suggesting that human enzyme may have a broader specificity than rat enzymes (46).

CORONARY HEART DISEASE AND RISK FACTORS

The influence of partially hydrogenated vegetable oils and trans fatty acids on serum cholesterol and lipoproteins has received great attention because these parameters are considered important risk factors associated with coronary heart disease (CHD). This subject presents with many disputed aspects and the data in the literature are open to different interpretations. A review of this topic was published very recently (47), where a large disagreement between different authors was be found. In fact some studies did not show either increased levels of total cholesterol and LDL-cholesterol or decreased amount of HDL-cholesterol; on the contrary, other investigations showed opposite results. These discrepancies are probably due to the different models used and to variables which include solid food versus liquid formula, percentage of trans out of the total lipids, amount of fats, carbohydrates, proteins, presence or absence of EFA, amount of cholesterol in the diet. As an example of the different results, in three controlled studies Mattson et al. (48) found similar plasma cholesterol concentration when elaldic acid was partially substituted for oleic acid; Mensink and Katan (49) showed that, at higher intakes of elaldic acid approximating those that prevail in the Netherlands, LDL cholesterol rose and HDL cholesterol fell; Laine et al. (50)

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concluded that, provided the linoleic acid content of the diet was high enough, partial hydrogenation of linoleic and oleic acid would not raise the plasma cholesterol level. It is worth while to note that two authoritative reports by the Food and Nutrition Board in the US (51) and by the British Nutrition Foundation (7) concluded that the effect of trans fatty acids on the cholesterol level was "neutral". In the Netherlands study, 11% energy was supplied from trans fatty acids, substantially more than the average intake in most industrialized countries and doubts have been raised as to whether those data could be extrapolated to populations with a lower level of intake (52). Because the implications of this controversy were considerable both for public health and for the edible oil industries, Nestel et al. (53) conducted a controlled double-blind study in which a more moderate amount of trans fatty acid was tested; in this study elaidic acid-rich diet was compared with an oleic acid-rich diet, as well as with two additional diets enriched with saturated fatty acids. The conclusion was that 3 weeks consumption of trans fatty acid (elaidic) gave LDL cholesterol level that did not differ from those seen with diet enriched with palmitic acid or butter fat but higher than when oleic acid was substituted for elaidic acid. These results are partially in agreement with the data of Zock et al. (54) demonstrating that 7.7% of energy (mean 24 g/day) of trans fatty acids in the diet significantly lowered HDL cholesterol and raised LDL cholesterol relative to linoleic acid. The combination of these data with those found by Mensink et al. (49) suggests a linear dose-response relationship between trans fatty acid intake and blood cholesterol. However replacement of linoleic acid by stearic acid also caused somewhat lower HDL and higher LDL cholesterol. Thus, based on the change in LDL/HDL ratio the trans diet had at least as much of an unfavorable effect as the saturated fat diet, suggesting that "the main health consequence of hydrogenating vegetable oils appears related to the reduction in linoleic and linolenic acids content rather than to the presence of trans and positional isomers (21). Personally I think that also the content of oleic acid should be taken into consideration. Moreover my personal opinion is that many of these studies should be regarded as "elegant" artificial laboratory experiments because the percentage of fats and simple sugars included in the tested diet are far removed from the recommended guide lines.

TRANS FATTY ACIDS AND Lp (a) SERUM CONCENTRATION To my opinion the important finding in the Nestel's paper was the observation that the elaidic acid-rich diet led to a significant elevation in the level of Lp(a) compared to all the other test diets including those with saturated fats and with palmitic acid. Whether trans mono unsaturated fatty acids permantly increase serum level of Lp(a) or not, remains an open question due to short time of the experiment. A comprehensive review on this lipoprotein was published recently (54). Briefly, lipoprotein (a) [Lp(a)] is a macromolecular complex resulting from the assembling of apoprotein B, cholesterol, phospho- and glycolipids and a protein called apo(a). This last one share sequence homology with plasminogen. Its blood concentration is largely under genetic ontrol and does not change much with age. l_~vels of Lp(a) which exceed 400 rag/1 are considered as one of the strong risk factor for CHD. Attempts to modify Lp(a) by drug or diet have not been very successful. The data of Nestel were subsequently confirmed by Mensink et al. (55) who

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used a diet containing 10% or 8% energy as trans fatty acids. The results obtained do suggest that the effects on Lp(a) concentration are proportional to the amount of trans fatty acids consumed.

CONCLUSIONS AND PROSPECTS The concerns about the possible physiometabolic influence of trans fatty acids raised in the early "/O's have "forced" oil refining industries to improve the technologies involved in margarine production; at present in fact, at least in Italy, the hydrogenation process was replaced by trans esterification resulting in a dramatic decrease of trans acids and related isomers. Moreover the consumption of this type of fats is far from the values found in other countries and the distribution of energy intake is more adherent to the up to date guide lines concerning, e.g., total fat intake. Personally I think that our diet is completely different from that reported as usual by Mensink and other authors previously cited. Given the complexity of fat metabolism it would seem prudent to avoid sweeping recommendations concerning the use of different fats, including partially hydrogenated fats, until results are obtained from experiments that are focused to answer specific questions. For these reason studies, based on reliable protocols, are needed to define the impact of hydrogenated vegetable oils on health and nutrition. However, in Italy, there is a lack of data concerning the estimated daily intake of geometrical and positional isomers derived from the consumption of foods prepared with shortenings or other foods like those indicated in Table 2. Lack of information on these products that can influence and/or interfere with lipid metabolism and membrane architecture, mainly in adolescents, is on the contrary a more important problem that needs to be addressed.

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